This invention relates to bioassays and more specifically to an antibody-based bioassay for enzyme-inducing chemicals.
Many xenobiotic compounds (compounds not normally found in an animal body) induce the formation in animal cells of enzymes which detoxify or modify such compounds. The results of the detoxification process, in most cases, is facilitated excretion of the offending compound A major source of xenobiotic modification in mammalian cells is the microsomal mixed-function oxidase system, a chain of enzymes terminating in one of a family of enzymes known as the cytochromes P-450. It is these terminal cytochromes that actually interact with the xenobiotic compound and chemically alter or metabolize it.
The spectrum of xenobiotic compounds which can be acted upon by the microsomal mixed-function oxidase system is extremely diverse. Studies designed to investigate the source of this diversity have demonstrated that it originates in the terminal cytochromes themselves. There are actually multiple forms of cytochrome P-450, each acting upon a group of loosely related chemical types. The form of cytochrome P-450 needed to interact with a xenobiotic compound is determined when that compound presents itself within the living cell. The cell responds to the presence of this foreign compound by increasing the synthesis of the appropriate cytochrome. This increase in cytochrome concentration in response to the presence of a specific chemical is referred to as induction.
The two forms of the terminal cytochrome relevant to this invention have been given the names cytochrome P.sub.1 -450, also known as alkyl hydrocarbon hydroxylase, and cytochrome P-448, otherwise referred to as aryl hydrocarbon hydroxylase. As used herein, the alkyl or aryl hydrocarbon hydroxylase designations refer to the type of enzymatic activity or substrate preferences associated with the induction of these enzymes. As also used herein, the abbreviation "AHH" will refer to either alkyl or aryl hydrocarbon hydroxylase, each of which is, in fact, a group of related enzymes.
In the presence of polycyclic aromatic hydrocarbons (aryl hydrocarbons) cytochrome P-448 concentration and activity increases in cells. When compounds such as the barbituates are present (hydrocarbons with less aryl or benzene-like character), cytochrome P.sub.1 -450 is increased. Thus increasing the induction of these enzymes by particular chemicals, for example by exposing cultured cells to samples containing various concentrations of one or the other types of inducers, provides a basis for detecting and measuring these chemicals in various kinds of samples.
The AHH enzymes are inducible enzymes, that is, their activity is increased in the presence of alkyl or aryl hydrocarbons. An AHH-inducing chemical rapidly penetrates the membrane of a living cell, and once within the cytoplasm, it interacts with a binding protein to form a complex. The complex is rapidly translocated into the nucleus of the cell, where it binds to a specific site on DNA. This binding serves as a signal for transcription of the adjacent DNA into messenger RNA that codes for AHH enzymes. This messenger RNA is rapidly translocated into the cell cytoplasm, where it is translated into AHH enzymes. The enzymes modify or detoxify toxic chemicals.
The complex also activates a second group of genes that code for proteins that control cell proliferation and differentiation. Activation of these genes can lead to cancerous transformation, a known property of many AHH inducers. Other AHH inducers are extremely toxic or harmful in other ways to living cells and organisms. Thus, measuring the induction of AHH by particular chemicals provides a basis for detecting and measuring the concentrations of these chemicals in various kinds of samples.
The level of AHH induction can be measured by measuring any of the AHH enzymes 7-Ethoxyresorufin-O-deethylase (EROD) one of the cytochrome P.sub.1 -450 enzymes, is conveniently used because the substrate, 7-ethoxyresorufin, is minimally toxic and the product, resorufin, can be measured spectrophotometrically at 572 nm according to the method of Klotz et al., Anal. Bioch., 140: 138-145 (1984), which is incorporated herein by reference, or fluorimetrically as described by Prough, et al. Meth. Enzymol., 52C: 372-377 (1978) which is incorporated herein by reference.
AHH inducers include the dibenzodioxins, dibenzofurans, and biphenyls. Polychlorinated dibenzodioxins have been the focus of much scientific and regulatory attention because of their extreme toxicity to experimental animals and their possible hazard to human beings. They occur as by-products and impurities in the production of chlorinated phenols and their conversion products, such as the phenoxy-acetic acid herbicides. The most toxic compound of this group known to date is 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD). Because of the toxicity of TCDD, it is desirable to detect it in the low parts per quadrillion range.
The present methods for detecting TCDD and related chemicals generally involve some form of low or high resolution chromatography, usually gas chromatagraphy, and high or low resolution mass spectrometry. (See Environmental Protection Agency Publication EPA/600/3-85/019, April 1985, entitled the National Dioxin Study.) This technology requires very expensive, sophisticated equipment, a high degree of skill on the part of the person running the assay, and a significant amount of time. Thus, it costs as much as $1,000-3,000 to test a sample for the presence of TCDD. In addition, this technique leaves much to be desired in terms of sensitivity. The lowest concentration of TCDD detectable is approximately 1 part per trillion.
Antibody detection techniques could significantly lower the cost and time associated with the detection of TCDD and similar chemicals. However, such techniques are limited by their sensitivity. One antibody technique, radioimmunoassay, has been proposed as a means of detecting TCDD in the low parts per trillion range Albro et al., Methods in Enzymology, 84: 619-628 (1982); Albro et al., Toxicol. Appl. Pharmacol., 50: 137-146 (1979). However, radioimmunoassay is still not sufficiently sensitive to detect concentrations of TCDD below 10 parts per trillion. It also requires careful handling because of the radioactivity and special procedures to dispose of the radioactive materials.
The presence of an AHH-inducing chemical in a sample can be determined by its ability to induce AHH in various types of cells. Once such cell culture assay is described in Bradlaw and Casterline, J. Assoc. Off. Anal. Chem., 62:904-916 (1979). The method is a cell culture-enzyme induction bioassay, which uses the induction of AHH activity in rat hepatoma cell line H4IIE to detect minute amounts of dibenzodioxins, dibenzofurans, and biphenyls. The authors state that the assay is a useful system for screening large numbers of specially prepared food extracts for contamination by these compounds, but that the system may not be able to specifically identify the reactive substance or substances. Thus, the system may be useful for identifying the presence of AHH-inducing chemicals in a sample, but it cannot be used to identify and quantify a particular AHH-inducing chemical, such as TCDD.